what they are Organelle Lights™ and Cellular Lights™ fluorescent protein–based reagents are targeted to subcellular compartments and organelles by signal sequences. Based on BacMam technology, these versatile tools are prepackaged in baculovirus particles for highly efficient, reproducible, and noncytopathic delivery to a range of cell types, including primary and stem cells. Organelle Lights™ and Cellular Lights™ fluorescent proteins are not overtly toxic, which ensures consistent expression. The efficient transduction makes it easy to deliver multiple constructs and to modulate gene expression. Organelle Lights™ and Cellular Lights™ fluorescent proteins are readily adaptable to a number of assay formats, and compatibility with automated liquid handling makes them ideal for high-content imaging applications. In addition, we now offer a red fluorescent protein (RFP): TagRFP from Evrogen. TagRFP is a novel monomeric red fluorescent protein based on the wild-type RFP from sea anemone Entacmaea quadricolor (Nat Methods (2007) 4:555–557). TagRFP is approximately three times brighter than mCherry protein (Nat Biotechnol (2004) 12:1567–1572), making it the brightest monomeric RFP currently available.

how they work Golgi-RFP: This new Organelle Lights™ product is TagRFP fused to a Golgi-targeting tag (a portion of human Golgi-resident enzyme N-acetylgalactosaminyltransferase-2). This is the same tag used in our popular Golgi-GFP.

Nuc-RFP: Our Organelle Lights™ Nuc-RFP comprises TagRFP fused to the SV40 nuclear localization sequence. This allows researchers to visualize the nucleus without using DNA-binding dyes, which may perturb cellular function.

H2B-GFP and -RFP: Another method of localizing the nucleus is via histone labeling. With the Cellular Lights™ H2B-GFP and -RFP products, researchers can localize histones in living cells and potentially follow these protein complexes during cell division.

Lysosomes-RFP: A new Organelle Lights™ target, Lysosomes-RFP is TagRFP fused to the lamp1 protein (lysosomal associated membrane protein 1). Researchers can now visualize lysosomes, important degradative organelles also involved in autolysis.

Endosomes-GFP: We have fused the bright and photostable Emerald GFP to Rab5a, an early endosomal marker, to produce another new Organelle Lights™ target. Endosomes-GFP will permit localization of the endosomes within the living cell, and can be fixed.

MAP4-GFP: One of our most exciting and visually stunning new Cellular Lights™ targets, MAP4 (microtubule associate protein 4) has been fused to Emerald GFP. This product will enable visualization of the microtubules within a living cell.

Tubulin-GFP and -RFP: Complementary to the Cellular Lights™ MAP4-GFP construct is our Cellular Lights™ Tubulin-GFP (Emerald GFP protein fusion) and -RFP (TagRFP protein fusion) fluorescent proteins. These two products are designed for live-cell imaging of microtubules.

Actin-GFP and -RFP: When added to mammalian cells, Actin-GFP and Actin-RFP Cellular Lights™ products will produce Emerald GFP or TagRFP fused to the N terminus of beta actin.

Null (control) virus: If the potential effects of baculovirus on your cell type are a concern, our new Cellular Lights™ Null virus, which lacks any mammalian genetic elements, provides a new alternative. The Null virus allows you to determine any possible baculovirus-based effects.

Aortic smooth muscle cells (Cascade Biologics, C-007-5C) were transduced with Cellular Lights™ MAP4-GFP and Lysosomes-RFP according to the established Molecular Probes® BacMam protocol. The following day Hoechst staining was performed, and cells were imaged using a 63x objective with GFP, RFP, and DAPI filters.

Human aortic smooth muscle cells (Cascade Biologics, C-007-5C) were transduced with Cellular Lights™ Tubulin-GFP according to the established Molecular Probes® BacMam protocol. The following day Hoechst staining was performed, and cells were imaged using a 63x objective with GFP and DAPI filters. Nocodazole treatment was performed (10 μM), and images obtained every 2.5 min for 15 min. Nocodazole is an antineoplastic agent that interferes with the polymerization of microtubules.

what they offer

intracellular landmarks that are well characterized, accurately targeted, safe, and easy to use—no need to make your own constructs and transfection complexes

what they are The WesternDot™ 625 Western Blot Kits combine the bright fluorescence properties of Qdot® 625 nanocrystals with the high-affinity streptavidin-biotin interaction to allow simple, highly sensitive detection of proteins immobilized on nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes. You get the sensitivity of chemiluminescence methods combined with brightness, photostability, flexibility, and greater ease of processing and imaging—no x-ray film, dark rooms, or developing reagents required.

how they work Incorporating a standard western blotting protocol, the detection step relies on a biotinylated secondary antibody, either goat anti-rabbit (W10132) or goat anti-mouse (W10142), followed by the key component, the Qdot® 625 streptavidin conjugate (Figure 1). The extremely high extinction coefficient of the Qdot® 625 nanocrystal in the UV and blue wavelengths combined with high quantum yield and an orange/red emission (Figure 2) allow for subnanogram sensitivity of protein detection using standard UV or blue-light–based detection systems. The fluorescent signal is compatible with the commonly used modes of fluorescence detection of DNA or protein gels and does not require specialized emission filters. Blots may be imaged wet or dry, and by epi- or transillumination.

Figure 2—Example of results achieved using the WesternDot™ 625 Western Blot Kit. Total proteins (2-fold dilution series ranging from 10 μg to ~10 ng) from Jurkat cell extract were analyzed on a NuPAGE® Novex® 4–12% Bis-Tris gel and then transferred to an Immobilon™-FL PVDF membrane. Immunodetection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous “housekeeping” protein in the Jurkat cell extract, was performed with the WesternDot™ 625 Goat Anti-Mouse Western Blot Kit using a mouse monoclonal anti-GAPDH antibody (Invitrogen Cat. no. 39-8600) at 1 μg/ml. The wet membrane was imaged using an Alpha Innotech HD2 instrument with a SYPRO® Red emission filter (620 +/– 40 nm) and excitation at 302 nm transillumination with an exposure time of 300 ms.

what it is Each Qdot® Antibody Conjugation Kit contains sufficient reagents, ultrafiltration columns, separation media, and associated components to perform two separate conjugation reactions. The protocol accompanying the kit details the method for conjugating Qdot® 625 nanocrystals to a 300 µg antibody sample, but the protocol can be adapted for conjugation to polyclonal Fab fragments and other classes of whole antibodies. Labeled antibodies can then be used in immunostaining (see figure) or other experiments requiring biofunctionalized labels (e.g., flow cytometry, ELISA).

how it works The Qdot® 625 Antibody Conjugation Kit (containing amine-derivatized, PEG-coated Qdot® nanocrystals and the amine–thiol cross linker SMCC) allows you to conjugate your own antibodies to our brightest visible dot with the peak of emission of 625 nm (see figure). The conjugation reaction can be completed in a few hours and is based on the fast and efficient coupling of thiols to reactive maleimide groups, which are present on the nanocrystals after SMCC activation. In addition to antibodies, other thiol-containing molecules can be coupled to the 625 nm nanocrystals using this kit.

The Qdot® 625 nanocrystal is characterized by a broad absorption spectrum and a narrow, symmetrical emission profile.

what it offers

the ultimate in fluorescence photostability and brightness

ease of use

flexibility to conjugate to most antibodies

compatibility with other Qdot® colors used with single-excitation sources for multispectral imaging with optimized filter sets

Learn more about Qdot® nanocrystal products. Also, be sure to investigate the BrightLine® QD625-A Filter Set—a new optimized filter set from Semrock, Inc. designed for use with Qdot® 625 nanocrystals.

The latest development in IgG antibody labeling—APEX Antibody Labeling Kits

what they are APEX Antibody Labeling Kits provide a convenient means to directly attach a fluorophore to very small amounts of IgG antibody (10–20 µg).

how they work These kits use a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX antibody labeling tip. This enables you to covalently label antibodies that are supplied in solutions containing stabilizing proteins, such as BSA or other contaminants, that can interfere with the amine-reactive labeling reagents used to attach the fluorophore to the antibody. Any contaminants are simply eluted through the tip. After the amine-reactive label is applied, a fluorescent IgG conjugate is ready for use in an imaging or flow cytometry application in as little as 2.5 hr with very little hands-on time.

what it is Dynabeads® FlowComp™ Flexi is an easy-to-use, flow-compatible kit for positive isolation of your cell type of choice by magnetic separation. This system is so flexible that almost any cell type can be isolated from any species and from a variety of samples—it depends only on the antibody you use. The isolated cells are ready to be used in any downstream application.

how it works The Dynabeads® FlowComp™ Flexi technology is simple: Through DSB-X™ biotinylation of antibodies and modified streptavidin on the Dynabeads®, you have the ideal platform to gently obtain bead-free cells in this tube-based cell isolation system. It's as simple as this:

what they are Invitrogen offers a number of primary antibodies against cell junction proteins, including tricellulin.

how they work Tight junctions form an important barrier of paracellular transport in epithelial cells, and the sealing of two adjacent cells at bicellular tight junctions is well described. The main structural components of bicellular tight junctions are claudins and occludins—tetra–membrane-spanning proteins. Less is known about the structure of tricellular tight junctions, a point where three adjacent cells are in contact with each other. Tricellulin is the first protein identified that specifically concentrates in tricellular tight junctions. Like claudins and occludins, tricellulin protein has four membrane-spanning domains, and it is highly expressed in epithelium-derived tissues such as small intestine, kidney, and lung.

what they offer

good specificity for imaging tricellular junctions

validated reagents with multiple applications and species specificities

part of a wide-ranging portfolio of antibodies for cell junction research

Localization of tricellular junctions using an antibody specific to the carboxy-terminus of tricellulin, in green (Cat. no. 48-8400). Bicellular tight junctions are revealed by the localization of occludin, in red (Cat. no. 33-1500).

what it is The immune system is regulated and maintained through apoptosis, a normal process in which cell populations are deleted in response to self-recognition, failure to bind MHC, and cytokine/growth factor withdrawal. To facilitate detection of apoptosis, we have developed a multiplex sandwich immunoassay that permits the simultaneous detection of three important biomarkers: cytochrome c (a protein that normally resides within the intermitochondrial space that is released to the cytosol in response to apoptotic stimuli), cleaved caspase-3 [175/176] (an important reporter for initiator caspase activation), and cleaved poly (ADP-ribose) polymerase (PARP) [214/215] (an important reporter for caspase-3 activation). how it works The Invitrogen™ Apoptosis 3-plex Panel is a solid-phase sandwich immunoassay that is designed for use with the Luminex® 100™ or 200™ instrument. Beads of defined spectral properties conjugated to analyte-specific capture antibodies, and samples (including standards of known analyte concentration, control specimens, and unknowns) are pipetted into the wells of a filter-bottom microplate and incubated for 2 hours. During this first incubation, analytes bind to the immobilized capture antibodies. After washing, a detector antibody is added to the mixture for 1 hour. At the end of this incubation, the beads are again washed and then incubated with an RPE conjugate (containing anti-rabbit RPE plus streptavidin RPE) for 30 minutes. During this final incubation, the RPE conjugate binds to the detector antibodies associated with the immune complexes on the beads, forming four-member solid-phase sandwiches. After washing to remove unbound RPE conjugate, the beads are analyzed with the Luminex® 100™ or 200™ instrument. By monitoring the spectral properties of the beads and the amount of associated RPE fluorescence, the concentrations of the three analytes can be determined.

what it is The Click-iT® EdU assay accurately measures proliferating cells (cells in S-phase) via the active uptake of the nucleoside analog EdU (5-ethynyl-2′-deoxyuridine) and eliminates the need for long incubations or harsh DNA denaturation and permeabilization procedures required by BrdU assays.

how it works Both the Click-iT® EdU and BrdU assays follow similar workflows. Cells or animals are given the nucleoside analog. Following fixation and permeabilization, the incorporated nucleoside is detected (see figure). Where EdU and BrdU methods differ is in their simplicity of detection; detection of EdU is based on a click reaction—a copper catalyzed covalent reaction between an azide and an alkyne. The small size of the dye azide allows for efficient detection of the incorporated EdU under mild conditions. Standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to gain access to the DNA. This is in contrast to the BrdU assays that require DNA denaturation (using HCl, heat, or DNAse) to expose the BrdU so that it may be detected with an anti-BrdU antibody which can require several hours to overnight incubation.

Proliferating cells labeled in vivo with the Click-iT® EdU system. EdU from the Click-iT® EdU Alexa Fluor® 488 Imaging Kit (C10083) was administered intraperitoneally to mice 2 hours prior to sacrifice. Intestinal tissues were formalin-fixed and paraffin-embedded. EdU was labeled using the Click-iT® reaction (~250 μl of reaction cocktail per slide). The tissue sections were washed and mounted in a medium containing DAPI. EdU-positive cells are labeled green, nuclei are stained with blue-fluorescent DAPI, and red autofluorescence was enhanced for image contrast. Image contributed by Sima Zacharek, Children's Hospital Boston.

what it offers DNA denaturation protocols using HCl and methanol can destroy cell morphology and antigen recognition sites. Although milder than HCl, DNase denaturation can destroy the ability to perform cell cycle analysis. Finding the balance between sufficient DNA denaturation and sufficient dsDNA amounts (required for cell cycle dye binding) is difficult. With Click-iT® EdU, content-rich results are now truly easy to obtain. You can not only accurately measure proliferation of individual cells by flow cytometry, microscopy, or high-throughput imaging (HCS), but also simultaneously detect cell cycle, intracellular, and extracellular targets in significantly less time than the BrdU method.

Can cell proliferation assays tell us more? Accurate assessment of the degree of DNA synthesis in proliferating cells provides important information in a wide range of pharmacological and regulatory studies. While the two methods currently used for measuring cell proliferation have given researchers useful insights, each method has its limitations. [
3H]-thymidine labeling is laborious and slow, offers poor resolution, and suffers from all the potential health and waste-disposal concerns inherent in radioligand methodology. BrdU labeling is faster and more sensitive, and offers better resolution; however, the success of this approach necessitates extensive sample denaturation that can be difficult to reproduce and can significantly degrade sample structure. In the present study, Salic and Mitchison demonstrate the advantages of "click" chemistry—incorporation of 5-ethynyl-2'-deoxyuridine (EdU) followed by labeling with a fluorescent azide derivative—as an alternative to existing cell proliferation assays. Their data show that EdU is extensively incorporated into the DNA of proliferating NIH 3T3, HeLa, and
Xenopus cells. Subsequent visualization of the fixed cells with Alexa Fluor® 488 or Alexa Fluor® 594 azide derivatives revealed efficient and highly reproducible labeling regardless of the Alexa Fluor® dye used. Comparison with BrdU staining carried out on the same cells revealed excellent colocalization of the signal from the two methods; however, the click methodology yielded substantially more intense fluorescence, with excellent preservation of sample structure. The authors further demonstrate that the click methodology can be used to visualize proliferation in unfixed cells, large tissue/organ explants, and even in live animals, greatly expanding the scope and utility of this already promising approach.

HaCaT cells (an immortalized human keratinocyte cell line) were seeded in an evaporation-free dish and allowed to grow to 80% confluency before being transfected with a mitochondria-targeted eGFP construct. Twenty-four hours after transfection, the cells were incubated for 15 min in a solution containing 1 μM Hoechst 33342 (
H21492) and then for 5 min in a fresh solution containing 8 μg/ml CellMask™ Deep Red stain ((
C10046); staining took place at 37°C in the presence of 5% CO
2). The final staining solution was removed and the sample was washed twice with DMEM (including 10% FBS). With the temperature maintained at a constant 37°C, a stack through the cell was imaged with a Zeiss AxioCam MRm camera and Axio Observer HS microscope fitted with a 100x oil alpha Plan-Apochromat objective and the BFP/GFP/HcRed filter set with triple emission/beam splitter and single excitation from Zeiss (62HE). The image stack was deconvolved with the Zeiss AxioVision 3D Deconvolution module. The point spread function was acquired using a 200 nm TetraSpeck™ fluorescent bead (
T7280) in water at 37°C. Metaphase chromosomes (pseudocolored blue, Hoechst 33342), plasma membrane (pseudocolored red, CellMask™ Deep Red stain), and mitochondria (pseudocolored green, eGFP) are all clearly visible. This image was one of a series of time-lapse images captured during a study aimed at tracking mitochondrial movements. The accumulated tracking data will be used to improve computer-based theoretical cell models being developed in cooperation with a group of theoretical physicists at the University of Saarland. Image submitted by Christian Junker, University of Saarland, Department of Biophysics, Homburg, Germany.

Signaling pathways Empower your research today with Invitrogen’s comprehensive portfolio of products and services for investigating cell signaling pathways—including high-quality reagents for basic research and assay development, validated biochemical and cell-based assays, and world-class profiling and screening services. See our portfolio of pathways at www.invitrogen.com/cellsignaling.

Choose the right dead-cell stain for flow cytometry Accurate interpretation of data obtained by flow cytometry relies on the correct identification and negation of the various nonspecific signals arising from dead cells in the population. Flow cytometrists at Invitrogen recently conducted a survey of 25 membrane stains that have been used to identify dead cells based on membrane integrity. They tested classic membrane-impermeant nucleic acid dyes, monomeric cyanine dyes, annexin V dyes, and amine-reactive dyes. All dyes were tested with a mixture of live and heat-killed cells, before and after formaldehyde fixation, with aged cultures, and with apoptotic cells. Their results demonstrate the utility of optimizing the concentration of dead-cell stain for each experiment and the importance of understanding how a particular dye performs in the intended application.

The Total Luminex® 200™ System Invitrogen has pioneered one of the most comprehensive analyte panels in the industry, and now offers the complete solution by introducing the Luminex® 200™ detection system. Experience the power of Luminex® multiplex analysis all from Invitrogen. The Total Luminex® 200™ System is a compact analyzer that performs up to 100 assays simultaneously in a single well of a microtiter plate. This system is a flexible analyzer based on the principles of flow cytometry, which integrates key xMAP® detection components such as lasers, optics, advanced fluidics, and high-speed digital signal processors. Utilizing the xPONENT® software of the xMAP® technology operating system, the Luminex® 200™ System offers optional technical controls for 21 CFR Part 11 compliance to provide an electronic audit trail.